Self-assembling-peptide-based structures and processes for controlling the self-assembly of such structures

ABSTRACT

The thermodynamics of self-assembling peptides may be altered to produce different morphologies. By altering environmental factors, initiation and propagation of self-assembly processes may be altered, thereby consequently altering the morphology of the resultant structure.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplications having Ser. No. 60/366,826, filed on Mar. 22, 2002, Ser.No. 60/420,746, filed on Oct. 23, 2003, and Express Mail mailing labelnumber EV269328445US, filed on Mar. 21, 2003, which are incorporatedherein by reference in their entireties.

FIELD OF INVENTION

The present invention relates generally to peptides and, moreparticularly, to self-assembling-peptide-based structures and processesfor controlling the self-assembly of such structures.

BACKGROUND

Nanotechnology has recently become of great interest for a variety ofreasons. For example, nanostructures may be used to generate devices ata molecular level, thereby permitting molecular-level probing.Specifically, it has been suggested that fibrils can be used forconnectors, wires, and actuators. Additionally, it has been suggestedthat nanotubes may be used as miniature pipettes for introducing smallproteins into biological or other systems.

Nanotubes may be generated through carefully controlled high-energykinetic processes, in which graphite-based structures (e.g., “bucky”tubes) are formed at extremely high temperatures. However, the outcomeof these kinetic processes is often difficult to predict, and theresulting structure tends to be heterogeneous.

Given the relatively unpredictable kinetic processes related tographite-based structures, a need exists in the industry for a robustnanostructure that can be created homogeneously and without the need forcomplicated kinetic processes.

SUMMARY

The present disclosure provides self-assembling-peptide-based structuresand processes for controlling the self-assembly of such structures.

Briefly described, in architecture, one embodiment is a fibril ornanotube structure generated as a result of controlling changes in theenvironment during a self-assembly process.

The present disclosure also provides processes for controlling theself-assembly of self-assembling-peptide-based structures.

In this regard, one embodiment of the method comprises the steps ofplacing a self-assembling peptide in a controlled environment, andcontrolling the initiation and propagation of a self-assembly process bycontrolling the environment.

Other systems, methods, features, and advantages will be or becomeapparent to one with skill in the art upon examination of the followingdrawings and detailed description. It is intended that all suchadditional systems, methods, features, and advantages be included withinthis description.

BRIEF DESCRIPTION OF THE DRAWINGS

Many aspects of the disclosure can be better understood with referenceto the following drawings. The components in the drawings are notnecessarily to scale, emphasis instead being placed upon clearlyillustrating the principles of the present invention. Moreover, in thedrawings, like reference numerals designate corresponding partsthroughout the several views.

FIG. 1 is a diagram illustrating the structure of an example amyloidfibril.

FIG. 2 is a diagram illustrating laminated β-sheets within the amyloidfibril of FIG. 1.

FIG. 3 is a diagram illustrating, in greater detail, two adjacentβ-sheets of FIG. 2 and the positions of the side chains.

FIG. 4 is a diagram illustrating potential metal ion binding sitesbetween two β-strands along the β-sheet within the laminated structureof FIG. 2.

FIG. 5A is a graph showing normalized rate of fibril formation as afunction of metal ion content for Aβ(10-21) (amino acid residues 10-21of SEQ ID NO: 1).

FIG. 5B is a graph showing normalized rate of fibril formation as afunction of metal ion content for Aβ(10-21)H13Q (SEQ ID NO: 3).

FIG. 6 is a diagram showing long homogeneous fibers that are formed inthe absence of metal ions.

FIG. 7 is a diagram showing numerous short fibers that are formed in thepresence of metal ions.

FIG. 8 is a diagram illustrating one embodiment of a structure as arectangular bilayer that is formed as an aggregate of fibril segments.

FIG. 9 is a graph showing progression of mean residue ellipticity overtime, which is indicative of the structures being formed over time.

FIG. 10 is a diagram illustrating a top view of an example nanotubeformed from amyloid fibrils.

FIG. 11 is an exploded view of a section of the nanotube of FIG. 10.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference is now made in detail to the description of the embodiments asillustrated in the drawings. While several embodiments are described inconnection with these drawings, there is no intent to limit theinvention to the embodiment or embodiments disclosed herein. On thecontrary, the intent is to cover all alternatives, modifications, andequivalents.

It is known that certain peptides self-assemble into variouspeptide-based structures through thermodynamic processes. Bymanipulating the environment of these self-assembling peptides, thenucleation and propagation of the self-assembly process may becontrolled. Consequently, manipulating environmental factors maypredictably control the morphology of the self-assembled structures. Theuse of self-assembling peptides is advantageous because, unlikegraphite-based structures that require high-energy kinetic processes,these self-assembling peptides organize themselves through thermodynamicprocesses. In this regard, the resulting peptide-based structurerequires very little overhead.

Another advantage in using self-assembling peptides is that, unlikelipid-based structures that are relatively loosely structured, thearchitectural integrity of the resulting peptide-based structure isfairly robust due to the hydrogen (H) bonds along the backbone thatdefine the structure. In this regard, peptide-based structures enjoy adistinct advantage over both lipid-based structures and graphite-basedstructures.

The description below provides methods for controlling nucleation andpropagation in forming peptide-based structures, thereby controlling themorphology of the resulting peptide-based structure. In a general sense,self-assembly provides a controlled environment where well-defined andhomogeneous structures form. The following description merely outlinesexample architectures that may be formed as a result of controllingenvironmental factors that affect the nucleation and propagation offormation.

FIG. 1 is a diagram illustrating the structure of an example amyloidfibril 10. Specifically, FIG. 1 shows an Aβ(10-35) (amino acid residues10-35 of SEQ ID NO: 1) fibril having multiple β-sheets that arelaminated and, in the aggregate, form the fibril 10. As shown in FIG. 1,hydrogen bonding (H-bonding) between adjacent residues results in alamination of multiple sheets. The H-bonding further stabilizes thestructure of the fibril 10. Depending on the length of the segments thatcomprise the fibril 10, the effect of the H-bonding between thesesegments may differ. Since the H-bonding contributes to the curvature ofthe fibril 10 as shown in FIG. 1, the length of the segments may furthercontribute to the degree of curvature of the fibril 10, thereby furtheraffecting the morphology of structures that can be formed from thefibrils. While the exact mechanism is still not fully understood, it isclear that topology is correlated to the length of the fiber segments.This is evidenced by the different resulting topologies from Aβ(10-35)(amino acid residues 10-35 of SEQ ID NO: 1) and Aβ(10-22) (amino acidresidues 16-22 of SEQ ID NO: 1), which are described in greater detailin the paper “Exploiting Amyloid Fibril Lamination for NanotubeSelf-Assembly,” by Lu et al., which is set forth fully in U.S.provisional patent application having Express Mail mailing label numberEV269328445US, filed on Mar. 21, 2003. Given this observation, it isclear that, in one embodiment, the architecture of self-assembledstructures may be altered by modifying the length of the component fibersegments.

The component structure of the amyloid fibril 10 is discussed in greaterdetail in FIGS. 2 and 3, which are diagrams illustrating laminatedβ-sheets 100 within the amyloid fibril 10 of FIG. 1. As shown in FIG. 2,the β-sheets 100 a . . . 100 f in Aβ(10-35) (amino acid residues 10-35of SEQ ID NO: 1) align parallel to each other due to the H-bonding.These sheets, as shown in FIG. 3, have a relatively fixed sheetseparation (D) that is governed by the attractive and repulsive forcesresulting from the formation of H-bonds along the backbone of the fibersegments. Additionally, residues 205 a . . . 205 d along the β-sheets100 g, 100 h are arranged in a fairly organized manner due to theseattractive and repulsive forces. For reasons provided below, theseresidues 205 a . . . 205 d may provide binding sites for substances suchas, for example, metal ions, which affect the nucleation and propagationof fibril formation.

Changing the acidity (pH) of the environment (e.g., betweenapproximately 2 and approximately 7.5) results in an alteration of theattractive and repulsive forces. Since, as described above, theself-assembling-peptide-based structure is likely based on H-bondsformed between the component segments, changes in the pH, which iseffectively an alteration of the H⁺ content, result in morphologicalchanges. Typically, the rate of formation decreases at lower pH andincreases at higher pH. In this regard, in another embodiment, theresulting morphology may be changed by altering the pH of theenvironment in which the self-assembly process takes place. Asnon-limiting examples, a pH of approximately 2.0 may provide arelatively homogeneous self-assembled structure while a more neutral pH(e.g., approximately 7.0 to approximately 7.4) may provide a fairlyheterogeneous self-assembled structure. The changes in morphology can beseen by comparing the different morphologies presented in the papers“Structure of the β-Amyloid(10-35) (amino acid residues 10-35 of SEQ IDNO: 1) Fibril,” by Burkoth et al., which is fully set forth in U.S.provisional patent application having Ser. No. 60/366,826, filed on Mar.22, 2002, “Metal Switch for Amyloid Formation: Insight into theStructure of the Nucleus,” by Morgan et al., which is fully set forth inU.S. provisional patent application 60/420,746, filed on Oct. 23, 2003,and “Exploiting Amyloid Fibril Lamination for Nanotube Self-Assembly.”

FIG. 4 is a diagram illustrating potential metal ion binding sitesbetween two β-strands in the laminated structure of FIG. 2. As shown inFIG. 4, the β-strands 120 a, 120 b have residues 305 a . . . 305 d thatare arranged in a particular configuration as a result of the H-bondswithin and between the β-strands 120 a, 120 b. Consequently, theresulting configuration produces potential binding sites in closeproximity to adjacent residues 305 a, 305 c between two β-strands 120 a,120 b that may bind to a metal ion 410 a. The changes in attractive andrepulsive forces due to the metal ion may further contribute to themorphology of the resulting peptide-based structure. Additionally, thepresence of the metal ions may facilitate the self-assembly process bypre-organizing the component segments. Thus, in another embodiment, thearchitecture of self-assembled structures may be altered by modifyingmetal content in the environment, thereby affecting how theself-assembling peptides interact with each other in forming theresulting self-assembled structure. Details related to the nucleationand propagation of self-assembly are described with reference to FIGS.5A and 5B, and are also described in the paper “Metal Switch for AmyloidFormation: Insight into the Structure of the Nucleus.”

FIG. 5A is a graph 500 showing normalized rate of fibril formation as afunction of metal ion content for Aβ(10-21) (amino acid residues 10-21of SEQ ID NO: 1). Specifically, FIG. 5A plots the normalized rate offibril formation on the y-axis 510 and the time on the x-axis 520 of thegraph 500. In the example of FIG. 5A, the metal ion is a zinc ion (Zn⁺²)which is introduced into the environment of the self-assembling peptideas zinc chloride (ZnCl₂). As shown in FIG. 5A, Aβ(10-21) (amino acidresidues 10-21 of SEQ ID NO: 1) self-assembles at a relatively slow ratein the absence of ZnCl₂. Comparatively, in the presence of ZnCl₂,Aβ(10-21) (amino acid residues 10-21 of SEQ ID NO: 1) self-assembles ata much higher rate to form the resulting self-assembled structure.

FIG. 5B is a graph 505 showing normalized rate of fibril formation as afunction of metal ion content for Aβ(10-21)H13Q (SEQ ID NO: 3), which isAβ(10-21) having a modified amino acid residue 13. Specifically, FIG. 5Bplots the normalized rate of fibril formation on the y-axis 510 and thetime on the x-axis 520 of the graph 505. Again, Zn⁺² is used as themetal ion. As shown in FIG. 5B, Aβ(10-21)H13Q (SEQ ID NO: 3) shows agreater propensity toward amyloid formation even in the absence of thezinc ion. Thus, for Aβ(10-21)H13Q (SEQ ID NO: 3), the nucleation periodwas greatly shortened so as to be almost undetected in FIG. 5B. As shownin FIG. 5B, the rate of self-assembly is comparatively higher forAβ(10-21)H13Q (SEQ ID NO: 3) in the presence of ZnCl₂ than in theabsence of ZnCl₂. While not shown in FIG. 5A or 5B, the nucleation (oractivation) of self-assembly is inhibited by the introduction of copper(Cu⁺²), rather than Zn⁺², into the environment of the self-assemblingpeptide.

Since the ramifications of FIGS. 5A and 5B are discussed in greaterdetail in the paper “Metal Switch for Amyloid Formation: Insight intothe Structure of the Nucleus,” only a truncated discussion of theeffects of ZnCl₂ on Aβ(10-21) (amino acid residues 10-21 of SEQ IDNO: 1) is discussed here. However, as evidenced by the two graphs 500,505, it should be appreciated that, in a more general sense, thepresence of metal ions affects the nucleation (or activation) andpropagation of the self-assembly process regardless of the exact peptidesequence. Additionally, as evidenced by FIGS. 5A and 5B, the nucleationand propagation of the self-assembly process may be altered by modifyingcertain segments of the peptide. In this regard, another embodiment ofthe process includes the step of altering segments within a peptide toaffect the nucleation and propagation of the self-assembly process.While FIGS. 5A and 5B show metal ions as specific nucleating elementsand inhibiting elements, it should be appreciated that other substancesmay be used as a nucleating element or inhibiting element. For example,as discussed with reference to the structure of the peptides, anysubstance that binds to a residue to affect the structure may be used asa nucleating element or an inhibiting element. Additionally, theinhibiting element may affect any of the self-assembly pathways that areundergone by the peptide during the self-assembly process. In thisregard, if the particular location and structure of the binding siteschanges as a function of time, then different stages of theself-assembly process may be inhibited or activated by such controllingsubstances. As non-limiting examples, other nucleating or inhibitingelements may include other metal ions, small organic molecules, designedpeptides and peptide analogs, nucleic acid analogs, or a combination ofthese elements.

As shown in FIGS. 3 and 4, since the metal ions likely bind at certainbinding sites along the peptide, the rate of formation may be a functionof the metal-ion-to-peptide concentration ratio. Thus, providing agreater metal-ion-to-peptide concentration ratio may more rapidlysaturate the binding sites with the metal ions. In this regard, inanother embodiment, changes in metal-ion-to-peptide concentration ratiosmay be altered to affect the resulting morphology. As non-limitingexamples, a higher metal-ion-to-peptide ratio may be approximately 1.5while a lower metal-ion-to-peptide ratio may be approximately 0.3. Also,since addition of metal ions also affects dielectric characteristics, inother embodiments, changes in the dielectric characteristics of thecontrolled environment may effect changes in morphology. While theaddition of metal ions illustrates changes in dielectriccharacteristics, it should be appreciated that the dielectriccharacteristics may be changed by other known techniques.

FIGS. 6 and 7 are diagrams showing different resulting fibers that areformed in the absence and presence of metal ions. Specifically, FIG. 6shows the resulting morphology in the absence of ZnCl₂ at an approximatepH of 2. As discussed above, in the absence of zinc ions, and at a lowerpH, the rate of assembly is relatively slow. Consequently, the slowformation of the self-assembled structures results in long heterogeneousfibers. Conversely, as shown in FIG. 7, in the presence of Zn⁺², thefaster rate of assembly results in numerous short fibers. Thus, FIGS. 6and 7 suggest that the presence of metal ions not only affects the rateof assembly (as shown in FIGS. 5A and 5B), but also affects thestability of the resulting structure.

FIG. 8 is a diagram illustrating one embodiment of a structure as arectangular bilayer 800 that is formed as an aggregate of fibrilsegments. Specifically, FIG. 8 shows a rectangular bilayer 800 formedusing Aβ(16-22) (amino acid residues 16-22 of SEQ ID NO: 1),CH₃CO-KLVFFAE-NH₂. As shown in FIG. 8, the Aβ(16-22) (amino acidresidues 16-22 of SEQ ID NO: 1) bilayer is approximately 130 nm wide by4 nm thick, with each leaflet being composed of β-sheets. Thecorresponding backbone H-bond is shown along the long axis 810 of therectangular bilayer 800, while the lamination is shown to progress alongthe 130-nm width of the rectangular bilayer 800. Since this rectangularbilayer 800 structure is described in greater detail in the paper“Exploiting Amyloid Fibril Lamination for Nanotube Self-Assembly,”further discussion of the rectangular bilayer 800 is omitted here.However, as further described below, the bilayer structure of FIG. 8 isused to further construct other architectures. In this regard, oneembodiment of a self-assembling-peptide-based structure may be seen as apeptide bilayer similar to that shown in FIG. 8.

FIG. 9 is a graph 900 showing progression of mean residue ellipticity930 over time, which is indicative of the structures being formed overtime. The mean residue ellipticity is plotted on the y-axis 910 whilethe time is plotted on the x-axis 920 of the graph 900. As shown in FIG.9, the mean residue ellipticity 930 shows that, after approximately 20hours, a negative ellipticity developed, which suggests the formation ofβ-sheets. Within the following 10 hours, the ellipticity changeddramatically, thereby suggesting the formation of helical ribbons, whichfurther progressed to nanotube structures. Since the progression of thestructures is discussed in greater detail in the paper “ExploitingAmyloid Fibril Lamination for Nanotube Self-Assembly,” only a truncateddiscussion is presented herein. It is worthwhile to note, however, thatthe resulting peptide-based nanotube structure is more robust and stablethan lipid-based nanotubes. Additionally, as shown in FIG. 9 and thepaper “Exploiting Amyloid Fibril Lamination for Nanotube Self-Assembly,”the peptide-based nanotubes are generated using a thermodynamic process,rather than a high-energy kinetic process as those required to generategraphite-based nanotubes.

FIG. 10 is a diagram illustrating a top view of an example nanotube 1000formed from Aβ(16-22) (amino acid residues 16-22 of SEQ ID NO: 1). Asshown in FIG. 10, and also in the paper “Exploiting Amyloid FibrilLamination for Nanotube Self-Assembly,” the nanotube 1000 has an innerradius of approximately 22 nm, an outer radius of approximately 26 nm,and a wall thickness (t) of approximately 4 nm. The wall thickness ofapproximately 4 nm is roughly twice the length of the Aβ(16-22) (aminoacid residues 16-22 of SEQ ID NO: 1) peptide, which suggests that thewall of the nanotube 1000 is composed of a peptide bilayer similar tothat shown in FIG. 8. This is shown in greater detail in FIG. 11.

FIG. 11 is an exploded view of a section of the nanotube 1000 defined bythe broken lines 1100 in FIG. 10. As shown in FIG. 11 and the paper“Exploiting Amyloid Fibril Lamination for Nanotube Self-Assembly,” theAβ(16-22) (amino acid residues 16-22 of SEQ ID NO: 1) peptide generatesa bilayer that is approximately 4 nm thick. The bilayer of FIG. 11 issimilar to the bilayer structure of FIG. 8 in that inner and outersurfaces of the bilayer are defined by β-sheets 110 i, 110 j, and eachparallel β-strand 120 c, 120 d is separated by a fixed separation (s)defined by the backbone H-bond. For the Aβ(16-22) (amino acid residues16-22 of SEQ ID NO: 1), the separation (s) is approximately 5 Å. Sincethe nanotube 1000 is discussed in greater detail in the paper“Exploiting Amyloid Fibril Lamination for Nanotube Self-Assembly,” onlya truncated discussion of the nanotube is presented here. However itshould be appreciated that the peptide-based nanotube 1000 results froma thermodynamic process, rather than a high-energy kinetic process thatis required for generation of graphite-based nanotubes, which results ina relatively low overhead. Additionally, unlike lipid-based structures,the peptide-based nanotube 1000 is fairly rigid and robust due to theH-bonds that, in part, define the structure.

In addition to forming such structures, the paper “Exploiting AmyloidFibril Lamination for Nanotube Self-Assembly” shows that the nanotubesmelt (or become unstable) at higher temperatures (e.g., approximately 80degrees Celsius). In this regard, for other embodiments, controllingtemperatures (e.g., maintaining a temperature less than approximately 80degrees Celsius) during the self-assembly process may be seen as oneapproach to controlling the morphology of a final self-assembledstructure.

As described with reference to FIGS. 1 through 11, and in the paper“Metal Switch for Amyloid Formation: Insight into the Structure of theNucleus,” the paper “Exploiting Amyloid Fibril Lamination for NanotubeSelf-Assembly,” and other papers included in the above-identifiedprovisional patent applications, the ability to manipulate theself-assembly process related to self-assembling peptides results in anovel approach to generating nanostructures. Additionally, bycontrolling the environment in which the self-assembling peptideundergoes the self-assembly process, the morphology of the resultingstructures may be altered. Furthermore, given the mechanisms thatunderlie the assembly of self-assembling peptides, these processes maybe activated and deactivated by controlling the environment in which theprocesses take place.

Although exemplary embodiments have been shown and described, it will beclear to those of ordinary skill in the art that a number of changes,modifications, or alterations may be made, none of which depart from thespirit of the present invention. For example, while specific peptideshave been illustrated above, it should be appreciated that variants ofthose disclosed embodiments are also within the scope of this invention.These variants may be generated by adding, deleting, or substituting atleast one amino acid, where the change may occur at the amino- orcarboxy-terminal positions of the reference peptide sequence or anywherebetween those terminal positions, interspersed either individually amongthe amino acids in the sequence or in one or more contiguous groupswithin the sequence, etc. Also, while not specifically discussed, itshould be appreciated that other environmental factors, such as, forexample, the media dielectric of the environment, may also be altered toeffect morphological changes. Moreover, while nanotubes, peptidebilayers, helices, long and short fibers, β-sheets, β-strands, etc. havebeen described above, it should be appreciated that the other structuresmay also be generated using the processes described above. Furthermore,in the context of nanostructures described herein, long fibers aredefined as any fiber having a fiber length that is greater than or equalto 500 nm, and short fibers are defined as those fibers having fiberlengths less than 500 nm. Additionally, while Aβ(10-21) (amino acidresidues 10-21 of SEQ ID NO: 1), Aβ(10-21)H13Q (SEQ ID NO: 3), andAβ(16-22) (amino acid residues 16-22 of SEQ ID NO: 1) have beenexplicitly discussed, it should be appreciated that the β-amyloidstructure may be Aβ(16-21) (amino acid residues 16-21 of SEQ ID NO: 1),Aβ(10-35) (amino acid residues 10-35 of SEQ ID NO: 1), Aβ(10-21)E11N(SEQ ID NO: 2), Aβ(1-40) (amino acid residues 1-40 of SEQ ID NO: 1),Aβ(1-42) (SEQ ID NO: 1;¹DAEFRHDSG¹⁰YEVHHQKLVFFAEDVGSNKGAIIGL³⁵MVGGVVI⁴²A), etc. All suchchanges, modifications, and alterations should therefore be seen aswithin the scope of the present invention.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, (i.e., peptide isosteres). “Polypeptide” refers to both shortchains (commonly referred to as peptides, oligopeptides, or oligomers)and to longer chains (generally referred to as proteins). “Polypeptides”may contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques, which are well known in the art. Suchmodifications are described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.

Modifications may occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentto the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched, and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination (Proteins—Structure and Molecular Properties, 2nd Ed., T.E. Creighton, W. H. Freeman and Company, New York, 1993; Wold, F.,Post-translational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in Post-translational Covalent Modification of Proteins, B. C.Johnson, Ed., Academic Press, New York, 1983; Seifter, et al., MethEnzymol, 182: 626-646, 1990, and Rattan, et al., Ann NY Acad. Sci.,663:48-62, 1992).

“Variant” refers to a polypeptide that differs from a referencepolypeptide, but retains essential properties. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,and deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polypeptide may be a naturally occurring such as an allelic variant,or it may be a variant that is not known to occur naturally.Non-naturally occurring variants of polypeptides may be made bymutagenesis techniques or by direct synthesis.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences as determined by comparing the sequences. In theart, “identity” also means the degree of sequence relatedness betweenpolypeptide sequences, as the case may be, as determined by the matchbetween strings of such sequences. “Identity” and “similarity” can bereadily calculated by known methods, including, but not limited to,those described in (Computational Molecular Biology, Lesk, A. M., Ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., Ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math.,48: 1073 (1988).

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs. Thepercent identity between two sequences can be determined by usinganalysis software (i.e., Sequence Analysis Software Package of theGenetics Computer Group, Madison Wis.) that incorporates the Needelmanand Wunsch, (J. Mol. Biol., 48: 443-453,1970) algorithm (e.g., NBLAST,and XBLAST). The default parameters are used to determine the identityfor the polypeptides of the present invention.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

Embodiments of the present invention also provide for amyloidpolypeptides that are substantially homologous to the amyloidpolypeptides of SEQ ID NO: 1. The term “substantially homologous” isused herein to denote polypeptides having about 50%, about 60%, about70%, about 80%, about 90%, and preferably about 95% sequence identity tothe sequences shown in SEQ ID NO: 1. Percent sequence identity isdetermined by conventional methods as discussed above.

In general, homologous polypeptides are characterized as having one ormore amino acid substitutions, deletions, and/or additions. Thesechanges are preferably of a minor nature, that is conservative aminoacid substitutions and other substitutions that do not significantlyaffect the activity of the polypeptide; small substitutions, typicallyof one to about six amino acids; and small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue, a small linkerpeptide of up to about 2-6 residues, or an affinity tag. Homologouspolypeptides comprising affinity tags can further comprise a proteolyticcleavage site between the homologous polypeptide and the affinity tag.

In addition, embodiments of the present invention include polypeptideshaving one or more “conservative amino acid substitutions,” comparedwith the amyloid polypeptide of SEQ ID NO: 1. Conservative amino acidsubstitutions can be based upon the chemical properties of the aminoacids. That is, variants can be obtained that contain one or more aminoacid substitutions of SEQ ID NO: 1, in which an alkyl amino acid issubstituted for an alkyl amino acid in a amyloid polypeptide, anaromatic amino acid is substituted for an aromatic amino acid in aamyloid polypeptide, a sulfur-containing amino acid is substituted for asulfur-containing amino acid in a amyloid polypeptide, ahydroxy-containing amino acid is substituted for a hydroxy-containingamino acid in a amyloid polypeptide, an acidic amino acid is substitutedfor an acidic amino acid in a amyloid polypeptide, a basic amino acid issubstituted for a basic amino acid in a amyloid polypeptide, or adibasic monocarboxylic amino acid is substituted for a dibasicmonocarboxylic amino acid in a amyloid polypeptide.

Amyloid polypeptides having conservative amino acid variants can alsocomprise non-naturally occurring amino acid residues. Non-naturallyoccurring amino acids include, without limitation,trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,trans-4-hydroxyproline, N-methyl-glycine, allo-threonine,methylthreonine, hydroxy-ethylcysteine, hydroxyethylhomocysteine,nitro-glutamine, homoglutamine, pipecolic acid, thiazolidine carboxylicacid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline,tert-leucine, norvaline, 2-azaphenyl-alanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenylalanine.

A limited number (i.e., less than 6) of non-conservative amino acids,amino acids that are not encoded by the genetic code, non-naturallyoccurring amino acids, and unnatural amino acids may be substituted foramyloid polypeptide amino acid residues.

Using the methods discussed herein, one of ordinary skill in the art canidentify and/or prepare a variety of amyloid polypeptide fragments orvariants of SEQ ID NO: 1 that retain the functional properties of theamyloid polypeptide.

Among the common amino acids, for example, a “conservative amino acidsubstitution” is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine andthreonine, (4) aspartate and glutamate, (5) glutamine and asparagine,and (6) lysine, arginine and histidine. Other conservative amino acidsubstitutions are provided in Table 1. TABLE 1 Characteristic Amino AcidBasic: arginine lysine histidine Acidic: glutamic acid aspartic acidPolar: glutamine asparagine Hydrophobic: leucine isoleucine valineAromatic: phenylalanine tryptophan tyrosine Small: glycine alanineserine threonine methionine

1. A process for controlling self-assembly ofself-assembling-peptide-based structures, the process comprising: (A)providing a controlled environment by: (A1) controlling content of metalions within the controlled environment, the metal ions being selectedfrom a group consisting of: (A1a) zinc ions; and (A1b) copper ions; (A2)controlling the acidity of the controlled environment, the acidity beingwithin the range of approximately pH 2.0 to approximately pH 7.4; (A3)controlling the temperature of the controlled environment, thetemperature being less than approximately 80 degrees Celsius; (A4)controlling the dielectric characteristics of the controlledenvironment; (A5) controlling a metal-ion-to-peptide concentration ratioin the controlled environment, the metal-ion-to-peptide concentrationratio being within the range of approximately 0.3 to approximately 1.5;(B) placing segments of β-amyloids in the controlled environment togenerate a self-assembling structure, (B 1) wherein the self-assemblingstructure is selected from a group consisting of: (B1a) a long fiberhaving a fiber length not less than approximately 500 nm; (B1b) a shortfiber having a fiber length less than approximately 500 nm; (B1c) ahelical structure; (B1d) a twisted ribbon structure; (B1e) a fibrillarstructure; (B1f) a peptide bilayer; and (B1g) a nanotube; (B2) whereinthe segment of the β-amyloid is selected from the group consisting of:(B2a) amino acid residues 10-21 of SEQ ID NO: 1 (Aβ(10-21)); (B2b) SEQID NO: 2 (Aβ(10-21)E11N); (B2c) SEQ ID NO: 3 (Aβ(10-21)H13Q); (B2d)amino acid residues 10-35 of SEQ ID NO: 1 (Aβ(10-35)); (B2e) amino acidresidues 16-21 of SEQ ID NO: 1 (Aβ(16-21)); (B2f) amino acid residues16-22 of SEQ ID NO: 1 (Aβ(16-22)); (B2g) amino acid residues 18-28 ofSEQ ID NO: 1 (Aβ(18-28)); (B2h) amino acid residues 1-40 of SEQ ID NO: 1(Aβ(1-40)); (B2i) SEQ ID NO: 1 (Aβ(1-42)).
 2. A process for controllingself-assembly of peptide-based structures, the process comprising:providing a controlled environment, the controlled environment beingadapted to redirect a self-assembly process, the self-assembly processbeing associated with a self-assembling peptide; and generating aself-assembling-peptide-based structure by placing the self-assemblingpeptide in the controlled environment.
 3. The process of claim 2,wherein the step of providing the controlled environment comprises:activating the self-assembly process by introducing a nucleatingelement.
 4. The process of claim 3, wherein the step of activating theself-assembly process comprises: introducing a metal ion.
 5. The processof claim 2, wherein the step of providing the controlled environmentcomprises: inhibiting a self-assembly pathway by introducing aninhibiting element.
 6. The process of claim 5, wherein the step ofinhibiting the self-assembly pathway comprises: introducing a metal ion.7. The process of claim 2, wherein the step of providing the controlledenvironment comprises a step selected from the group consisting of:controlling content of nucleating elements within the controlledenvironment; and controlling content of inhibiting elements within thecontrolled environment.
 8. The process of claim 7, wherein the step ofcontrolling the content of nucleating elements comprises: controllingcontent of zinc ions within the controlled environment.
 9. The processof claim 7, wherein the step of controlling the content of inhibitingelements comprises: controlling content of copper ions within thecontrolled environment.
 10. The process of claim 2, wherein the step ofproviding the controlled environment comprises a step selected from thegroup consisting of: controlling a nucleating-element-to-peptideconcentration ratio within the controlled environment; and controllingan inhibiting-element-to-peptide concentration ratio within thecontrolled environment.
 11. The process of claim 2, wherein the step ofproviding the controlled environment comprises: controlling the acidityof the controlled environment.
 12. The process of claim 2, wherein thestep of providing the controlled environment comprises: controlling thetemperature of the controlled environment.
 13. The process of claim 2,wherein the step of providing the controlled environment comprises:controlling the dielectric characteristics of the controlledenvironment.
 14. The process of claim 2, wherein the step of generatingthe self-assembling structure comprises: generating a long fiber havinga fiber length not less than approximately 500 nm.
 15. The process ofclaim 2, wherein the step of generating the self-assembling structurecomprises: generating a short fiber having a fiber length less thanapproximately 500 nm.
 16. The process of claim 2, wherein the step ofgenerating the self-assembling structure comprises: generating a helicalstructure.
 17. The process of claim 2, wherein the step of generatingthe self-assembling structure comprises: generating a peptide bilayer.18. The process of claim 2, wherein the step of generating theself-assembling structure comprises: generating a nanotube.
 19. Theprocess of claim 2, wherein the step of generating the self-assemblingstructure comprises: placing a segment of a β-amyloid in the controlledenvironment, wherein the segment of the β-amyloid is selected from agroup consisting of: amino acid residues 10-21 of SEQ ID NO: 1)(Aβ(10-21) and variants thereof; SEQ ID NO: 2 (Aβ(10-21)E11N) andvariants thereof; SEQ ID NO: 3 (Aβ(10-21)H13Q) and variants thereof;amino acid residues 16-21 of SEQ ID NO: 1 (Aβ(16-21)) and variantsthereof; amino acid residues 16-22 of SEQ ID NO: 1 (Aβ(16-22)) andvariants thereof.
 20. The process of claim 2, wherein the step ofgenerating the self-assembling structure comprises: placing a segment ofa β-amyloid in the controlled environment, wherein the segment of theβ-amyloid is selected from a group consisting of: amino acid residues10-35 of SEQ ID NO: 1 (Aβ(10-35)) and variants thereof; amino acidresidues 18-28 of SEQ ID NO: 1 (Aβ(18-28)) and variants thereof; aminoacid residues 1-40 of SEQ ID NO: 1 (Aβ(1-40)) and variants thereof; andSEQ ID NO: 1 (Aβ(1-42)) and variants thereof.
 21. A process forcontrolling self-assembly of self-assembling-peptide-based structures,the process comprising: placing a self-assembling peptide in acontrolled environment; controlling initiation of a self-assemblyprocess, the self-assembly process being associated with theself-assembling peptide; and controlling propagation of theself-assembly process.
 22. The process of claim 21, wherein the step ofplacing the self-assembling peptide in the controlled environmentcomprises: placing a segment of a β-amyloid in the controlledenvironment, wherein the segment of the β-amyloid is selected from agroup consisting of: amino acid residues 10-21 of SEQ ID NO: 1(Aβ(10-21) and variants thereof; SEQ ID NO: 2 (Aβ(10-21)E11N) andvariants thereof; SEQ ID NO: 3 (Aβ(10-21)H13Q) and variants thereof;amino acid residues 16-21 of SEQ ID NO: 1 (Aβ(16-21)) and variantsthereof; amino acid residues 16-22 of SEQ ID NO: 1 (Aβ(16-22)) andvariants thereof.
 23. The process of claim 21, wherein the step ofplacing the self-assembling peptide in the controlled environmentcomprises: placing a segment of a β-amyloid in the controlledenvironment, wherein the segment of the β-amyloid is selected from agroup consisting of: amino acid residues 10-35 of SEQ ID NO: 1(Aβ(10-35)) and variants thereof; amino acid residues 18-28 of SEQ IDNO: 1 (Aβ(18-28)) and variants thereof; amino acid residues 1-40 of SEQID NO: 1 (Aβ(1-40)) and variants thereof; SEQ ID NO: 1 (Aβ(1-42)) andvariants thereof.
 24. The process of claim 21, wherein the step ofcontrolling initiation of the self-assembly process comprises:activating the self-assembly process by adding a nucleating element. 25.The process of claim 21, wherein the step of controlling initiation ofthe self-assembly process comprises: inhibiting the self-assemblyprocess by adding an inhibiting element.
 26. The process of claim 21,wherein the step of controlling initiation of the self-assembly processcomprises a step selected from the group consisting of: controllingcontent of nucleating elements within the controlled environment; andcontrolling content of inhibiting elements within the controlledenvironment.
 27. The process of claim 21, wherein the step ofcontrolling initiation of the self-assembly process comprises a stepselected from the group consisting of: controlling anucleation-element-to-peptide concentration ratio in the controlledenvironment; and controlling an inhibiting-element-to-peptideconcentration ratio in the controlled environment.
 28. The process ofclaim 21, wherein the step of controlling initiation of theself-assembly process comprises: controlling the temperature of thecontrolled environment.
 29. The process of claim 21, wherein the step ofcontrolling propagation of the self-assembly process comprises:controlling content of metal ions within the controlled environment. 30.The process of claim 21, wherein the step of controlling propagation ofthe self-assembly process comprises: controlling a metal-ion-to-peptideconcentration ratio in the controlled environment.
 31. The process ofclaim 21, wherein the step of controlling propagation of theself-assembly process comprises: controlling the temperature of thecontrolled environment.
 32. A self-assembling-peptide-based structurecomprising: segments of a β-amyloid, the segments being selected from agroup consisting of: amino acid residues 10-21 of SEQ ID NO: 1(Aβ(10-21); SEQ ID NO: 2 (Aβ(10-21)E11N); SEQ ID NO: 3 (Aβ(10-21)H13Q);amino acid residues 16-21 of SEQ ID NO: 1 (Aβ(16-21)); amino acidresidues 16-22 of SEQ ID NO: 1 (Aβ(16-22)); amino acid residues 10-21 ofSEQ ID NO: 1 (Aβ(10-21) with a conservative amino acid substitution; SEQID NO: 2 (Aβ(10-21)E11N) with a conservative amino acid substitution;SEQ ID NO: 3 (Aβ(10-21)H13Q) with a conservative amino acidsubstitution; amino acid residues 16-21 of SEQ ID NO: 1 (Aβ(16-21)) witha conservative amino acid substitution; and amino acid residues 16-22 ofSEQ ID NO: 1 (Aβ(16-22)) with a conservative amino acid substitution;and hydrogen bonds formed between the segments of the β-amyloid.
 33. Theself-assembling-peptide-based structure of claim 32, wherein thestructure is a long fiber having a fiber length not less thanapproximately 500 nm.
 34. The self-assembling-peptide-based structure ofclaim 32, wherein the structure is a short fiber having a fiber lengthless than approximately 500 nm.
 35. The self-assembling-peptide-basedstructure of claim 32, wherein the structure is a peptide bilayer. 36.The self-assembling-peptide-based structure of claim 32, wherein thestructure is a fibrillar structure.
 37. Theself-assembling-peptide-based structure of claim 32, wherein thestructure is a helical structure.
 38. The self-assembling-peptide-basedstructure of claim 32, wherein the structure is a twisted ribbonstructure.
 39. The self-assembling-peptide-based structure of claim 32,wherein the structure is a nanotube.
 40. Theself-assembling-peptide-based structure of claim 39, wherein thenanotube comprises: a wall thickness of approximately 4 nm; and an outerdiameter between approximately 50 nm and approximately 100 nm.
 41. Theself-assembling-peptide-based structure of claim 32, wherein thestructure is a peptide bilayer having: a thickness of approximately 4nm; and a width of approximately 130 nm.
 42. Aself-assembling-peptide-based structure comprising: self-assemblingpeptides; and hydrogen bonds formed between self-assembling peptides toform a nanotube.
 43. The self-assembling-peptide-based structure ofclaim 42, wherein the nanotube comprises: a wall thickness ofapproximately 4 nm; and an outer diameter between approximately 50 nmand approximately 100 nm.